The invention relates to nucleic acid sequences isolated from sugarcane and to methods of using them. In particular, the inventions relates to nucleotide sequences which are derived from sugarcane polyubiquitin genes and which are capable of directing constitutive expression of a nucleic acid sequence of interest that is operably linked to the sugarcane polyubiquitin nucleotide sequences. The sugarcane polyubiquitin nucleotide sequences are useful in regulating expression of a nucleic acid sequence of interest in monocotyledonous and dicotyledonous plants.
Much scientific effort has been directed at genetically engineering plants to produce agronomically relevant proteins. Recombinant genes for producing proteins in plants require a promoter sequence which is capable of directing protein expression in plant cells. Promoter sequences which direct high levels of protein expression in plant cells are particularly desirable since fewer numbers of transgenic plants need to be produced and screened to recover plants producing agronomically significant quantities of the target protein. In addition, high levels of protein expression aid the generation of plants which exhibit commercially important phenotypic properties, such as pest and disease resistance, resistance to environmental stress (e.g., water-logging, drought, heat, cold, light-intensity, day-length, chemicals, etc.), improved qualities (e.g., high yield of fruit, extended shelf-life, uniform fruit shape and color, higher sugar content, higher vitamins C and A content, lower acidity, etc.).
Some promoter sequences which are capable of driving expression of transgenes (e.g., selectable marker genes) in plants are known in the art and are derived from a variety of sources such as bacteria, plant DNA viruses, and plants. Promoters of bacterial origin include the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from native Ti plasmids. Promoters of viral origin include the 35S and 19S RNA promoters of cauliflower mosaic virus. Plant promoters include the ribulose-1,3-diphosphate carboxylase small subunit promoter, the phaseolin promoter, and the maize polyubiquitin promoter.
While some promoter sequences which function in plant cells are available, expression of more than one gene (e.g., a selectable marker gene and an agronomically relevant gene) which is operably linked to the same promoter sequence is likely to be hampered by homology dependent silencing of transgenes in plants. Thus, what is needed are additional promoter sequences which are capable of driving transgene expression. In particular, what is needed are promoter sequences which drive transgene expression in both monocotyledonous and dicotyledonous plant cells.
The present invention provides nucleic acid sequences having promoter activity. The nucleic acid sequences provided herein direct expression of operably linked nucleotide sequences in cells, tissues and organs of monocotyledonous and dicotyledonous plants. In one embodiment, the invention provides a substantially purified nucleic acid sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:7, the complement of SEQ ID NO:7, homologs of SEQ ID NO:7, homologs of the complement of SEQ ID NO:7; SEQ ID NO:10, the complement of SEQ ID NO:10, homologs of SEQ ID NO:10, and homologs of the complement of SEQ ID NO:10. In a preferred embodiment, the nucleotide sequence is characterized by having promoter activity. In a more preferred embodiment, the promoter activity is constitutive. In another embodiment, nucleotide sequence is double-stranded. In yet another embodiment, the nucleotide sequence is single-stranded. In yet another alternative embodiment, the nucleic acid sequence is contained in a plant cell. In a preferred embodiment, the plant cell is derived from a monocotyledonous plant. In a more preferred embodiment, the monocotyledonous plant is selected from the group consisting of sugarcane, maize, sorghum, pineapple, rice, barley, oat, wheat, rye, yam, onion, banana, coconut, date, and hop. In an alternative more preferred embodiment, the plant cell is derived from a dicotyledonous plant. In a preferred embodiment, the dicotyledonous plant is selected from the group consisting of tobacco, tomato, soybean, and papaya.
The invention also provides a substantially purified nucleic acid sequence comprising a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO:7 and the complement thereof. In a preferred embodiment, the portion is characterized by having promoter activity. In a more preferred embodiment, the promoter activity is constitutive. In an alternative preferred embodiment, the portion is double-stranded. In another alternative preferred embodiment, the portion is single-stranded. In yet another alternative preferred embodiment, the portion comprises the nucleotide sequence selected from the group consisting of the nucleotides from 1 to 242, from 245 to 787, from 788 to 1020, from 1021 to 1084, from 1085 to 1168, from 1169 to 1173, from 1174 to 1648, from 1649 to 1802, from 1 to 377, from 378 to 442, and from 443 to 1802. In a further alternative preferred embodiment, nucleic acid sequence is contained in a plant cell. In a more preferred embodiment, the plant cell is derived from a monocotyledonous plant. In a yet more preferred embodiment, the monocotyledonous plant is selected from the group consisting of sugarcane, maize, sorghum, pineapple, rice, barley, oat, wheat, rye, yam, onion, banana, coconut, date, and hop. In another more preferred embodiment, the plant cell is derived from a dicotyledonous plant. In a yet more preferred embodiment, the dicotyledonous plant is selected from the group consisting of tobacco, tomato, soybean, and papaya.
Further provided by the invention is a substantially purified nucleic acid sequence comprising a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO:10 and the complement thereof. In a preferred embodiment, the portion is characterized by having promoter activity. In a more preferred embodiment, the promoter activity is constitutive. In an alternative preferred embodiment, the portion is double-stranded. In another alternative preferred embodiment, the portion is single-stranded. In yet another alternative preferred embodiment, the portion comprises the nucleotide sequence selected from the group consisting of the nucleotides 1 to 3600, from 3602 to 3612, from 3614 to 3688, from 1 to 2248, from 2249 to 2313, from 2314 to 3688, and from 1671 to 2248. In another alternative preferred embodiment, the nucleic acid sequence is contained in a plant cell. In a more preferred embodiment, the plant cell is derived from a monocotyledonous plant. In yet a more preferred embodiment, the monocotyledonous plant is selected from the group consisting of sugarcane, maize, sorghum, pineapple, rice, barley, oat, wheat, rye, yam, onion, banana, coconut, date, and hop. In an alternative more preferred embodiment, the plant cell is derived from a dicotyledonous plant. In a yet more preferred embodiment, the dicotyledonous plant is selected from the group consisting of tobacco, tomato, soybean, and papaya.
The invention additionally provides a substantially purified nucleic acid sequence comprising the EcoRI/XbaI fragment isolated from plasmid pubi4-GUS contained in Escherichia coli cells deposited as NRRLB-30115, the complement of the fragment, homologs of the fragment, and homologs of the complement of the fragment. In a preferred embodiment, the nucleotide sequence is SEQ ID NO:7. In a more preferred embodiment, the nucleotide sequence is characterized by having promoter activity. In a yet more preferred embodiment, the promoter activity is constitutive. In an alternative yet more preferred embodiment, the nucleotide sequence is double-stranded. In another alternative more preferred embodiment, the nucleotide sequence is single-stranded. In yet another alternative more preferred embodiment, the nucleic acid sequence is contained in a plant cell. In another preferred embodiment, the plant cell is derived from a monocotyledonous plant. In a more preferred embodiment, the monocotyledonous plant is selected from the group consisting of sugarcane, maize, sorghum, pineapple, rice, barley, oat, wheat, rye, yam, onion, banana, coconut, date, and hop. In an alternative preferred embodiment, the plant cell is derived from a dicotyledonous plant. In a yet more preferred embodiment, the dicotyledonous plant is selected from the group consisting of tobacco, tomato, soybean, and papaya.
Also provided herein is a substantially purified nucleic acid sequence comprising the HindIII/XbaI fragment isolated from plasmid pubi9-GUS contained in Escherichia coli cells deposited as NRRLB-30116, the complement of the fragment, homologs of the fragment, and homologs of the complement of the fragment. In a preferred embodiment, the nucleotide sequence is SEQ ID NO:10. In an alternative preferred embodiment, the nucleotide sequence is characterized by having promoter activity. In a more preferred embodiment, the promoter activity is constitutive. In another alternative preferred embodiment, the nucleotide sequence is double-stranded. In yet another alternative preferred embodiment the nucleotide sequence is single-stranded. In another alternative preferred embodiment, the nucleic acid sequence is contained in a plant cell.In a more preferred embodiment, the plant cell is derived from a monocotyledonous plant. In a yet more preferred embodiment, the monocotyledonous plant is selected from the group consisting of sugarcane, maize, sorghum, pineapple, rice, barley, oat, wheat, rye, yam, onion, banana, coconut, date, and hop. In another more preferred embodiment, the plant cell is derived from a dicotyledonous plant. In a yet more preferred embodiment, the dicotyledonous plant is selected from the group consisting of tobacco, tomato, soybean, and papaya.
The invention further provides a substantially purified nucleic acid sequence comprising a portion of the EcoRI/XbaI fragment isolated from plasmid pubi4-GUS contained in Escherichia coli cells deposited as NRRLB-30115, and the complement of the fragment. In a preferred embodiment, the portion is characterized by having promoter activity. In a more preferred embodiment, the promoter activity is constitutive. In an alternative preferred embodiment, the portion is double-stranded. In another alterative preferred embodiment, the portion is single-stranded. In yet another alternative preferred embodiment, the nucleic acid sequence is contained in a plant cell. In a more preferred embodiment, the plant cell is derived from a monocotyledonous plant.In a yet more preferred embodiment, the monocotyledonous plant is selected from the group consisting of sugarcane, maize, sorghum, pineapple, rice, barley, oat, wheat, rye, yam, onion, banana, coconut, date, and hop. In an alternative more preferred embodiment, the plant cell is derived from a dicotyledonous plant. In yet a more preferred embodiment, the dicotyledonous plant is selected from the group consisting of tobacco, tomato, soybean, and papaya.
Also provided by the invention is a substantially purified nucleic acid sequence comprising a portion of the HindIII/XbaI fragment isolated from plasmid pubi9-GUS contained in Escherichia coli cells deposited as NRRLB-30116, and the complement of the fragment. In a preferred embodiment, the portion is characterized by having promoter activity. In a more preferred embodiment, the promoter activity is constitutive. In another preferred embodiment, the portion is double-stranded. In yet another preferred embodiment, the portion is single-stranded. In yet another preferred embodiment, the nucleic acid sequence is contained in a plant cell. In a more preferred embodiment, the plant cell is derived from a monocotyledonous plant. In a yet more preferred embodiment, the monocotyledonous plant is selected from the group consisting of sugarcane, maize, sorghum, pineapple, rice, barley, oat, wheat, rye, yam, onion, banana, coconut, date, and hop. In an alterative more preferred embodiment, the plant cell is derived from a dicotyledonous plant. In a yet more preferred embodiment, the dicotyledonous plant is selected from the group consisting of tobacco, tomato, soybean, and papaya.
The invention additionally provides a recombinant expression vector comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:7, the complement of SEQ ID NO:7, homologs of SEQ ID NO:7, homologs of the complement of SEQ ID NO:7; SEQ ID NO:10, the complement of SEQ ID NO:10, homologs of SEQ ID NO:10, and homologs of the complement of SEQ ID NO:10. In a preferred embodiment, the recombinant expression vector is selected from the group consisting of pubi4-GUS, pubi9-GUS, 4PI-GUS and 9PI-GUS.
The invention also provides a recombinant expression vector comprising a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO:7 and the complement thereof.
Also provided herein is a recombinant expression vector comprising a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO:10 and the complement thereof.
Additionally provided by the invention is a transgenic plant cell comprising a nucleic acid sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:7, the complement of SEQ ID NO:7, homologs of SEQ ID NO:7, homologs of the complement of SEQ ID NO:7; SEQ ID NO:10, the complement of SEQ ID NO:10, homologs of SEQ ID NO:10, and homologs of the complement of SEQ ID NO:10, wherein the nucleotide sequence is operably linked to a nucleic acid sequence of interest. In a preferred embodiment, the transgenic plant cell expresses the nucleic acid sequence of interest. In a more preferred embodiment, the expression is constitutive. In an alternative embodiment, the transgenic plant cell is derived from a monocotyledonous plant. In a more preferred embodiment, the monocotyledonous plant is selected from the group consisting of sugarcane, maize, sorghum, pineapple, rice, barley, oat, wheat, rye, yam, onion, banana, coconut, date, and hop. In another alternative embodiment, the transgenic plant cell is derived from a dicotyledonous plant. In a more preferred embodiment, the dicotyledonous plant is selected from the group consisting of tobacco, tomato, soybean, and papaya. In yet another alternative embodiment, the nucleic acid sequence of interest is a sense sequence. In a more preferred embodiment, the sense sequence encodes a protein selected from the group consisting of xcex2-glucuronidase, luciferase, xcex2-galactosidase, 1-aminocyclopropane-1-carboxylic acid deaminase, sucrose phosphate synthase, 5-enolpyruvyl-3- phosphoshikimate synthase, acetolactate synthase, RNase, wheat germ agglutinin, sweetness protein, and Bacillus thuringiensis crystal toxin proteins. In a further alternative embodiment, the nucleic acid sequence of interest is an antisense sequence. In a more preferred embodiment, the antisense sequence is selected from the group consisting of an antisense sequence to ACC synthase, to ethylene inducible sequences, and to polyphenol oxidase.
The invention further provides a transgenic plant cell comprising a nucleic acid sequence comprising a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO:7 and the complement thereof.
Also provided herein is a transgenic plant cell comprising a nucleic acid sequence comprising a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO:10 and the complement thereof.
Additionally provided by the invention is a method for expressing a nucleic acid sequence of interest in a plant cell, comprising: a) providing: i) a plant cell; ii) a nucleic acid sequence of interest; and iii) a nucleotide sequence selected from the group consisting of SEQ ID NO:7, the complement of SEQ ID NO:7, homologs of SEQ ID NO:7, homologs of the complement of SEQ ID NO:7; SEQ ID NO:10, the complement of SEQ ID NO:10, homologs of SEQ ID NO:10, and homologs of the complement of SEQ ID NO:10; b) operably linking the nucleic acid sequence of interest to the nucleotide sequence to produce a transgene; and c) introducing the transgene into the plant cell to produce a transgenic plant cell under conditions such that the nucleic acid sequence of interest is expressed in the transgenic plant cell. In a preferred embodiment, the method further comprises d) identifying the transgenic plant cell. In another preferred embodiment, the method further comprises d) regenerating transgenic plant tissue from the transgenic plant cell. In an alternative preferred embodiment, the methods further comprises d) regenerating a transgenic plant from the transgenic plant cell. In another preferred embodiment, the plant cell is derived from a monocotyledonous plant. In yet a more preferred embodiment, the monocotyledonous plant is selected from the group consisting of sugarcane, maize, sorghum, pineapple, rice, barley, oat, wheat, rye, yam, onion, banana, coconut, date, and hop. In another alterative more preferred embodiment the plant cell is derived from a dicotyledonous plant. In a yet more preferred embodiment, the dicotyledonous plant is selected from the group consisting of tobacco, tomato, soybean, and papaya.
The invention further provides a method for expressing a nucleic acid sequence of interest in a plant cell, comprising: a) providing: i) a plant cell; ii) a nucleic acid sequence of interest; and iii) a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO:7 and the complement thereof; b) operably linking the nucleic acid sequence of interest to the portion of a nucleotide sequence to produce a transgene; and c) introducing the transgene into the plant cell to produce a transgenic plant cell under conditions such that the nucleic acid sequence of interest is expressed in the transgenic plant cell. In a preferred embodiment, the portion comprises the nucleotide sequence selected from the group consisting of the nucleotides from 1 to 242, from 245 to 787, from 788 to 1020, from 1021 to 1084, from 1085 to 1168, from 1169 to 1173, from 1174 to 1648, from 1649 to 1802, from 12 to 377, from 378 to 442, and from 443 to 1802.
Also provided by the invention is a method for expressing a nucleic acid sequence of interest in a plant cell, comprising: a) providing: i) a plant cell; ii) a nucleic acid sequence of interest; and iii) a portion of a nucleotide sequence selected from the group consisting of SEQ ID NO:10 and the complement thereof; b) operably linking the nucleic acid sequence of interest to the portion of a nucleotide sequence to produce a transgene; and c) introducing the transgene into the plant cell to produce a transgenic plant cell under conditions such that the nucleic acid sequence of interest is expressed in the transgenic plant cell. In a preferred embodiment, the portion comprises the nucleotide sequence selected from the group consisting of the nucleotides 1 to 3600, from 3602 to 3612, from 3614 to 3688, from 1 to 2248, from 2249 to 2313, from 2314 to 3688, and from 1671 to 2248.